Myxococcus xanthus uses cell-cell interactions to coordinate a developmental program in which cells aggregate to form organized multicellular fruiting bodies and differentiate from rod-shaped cells into ovoid spores. This developmental program is initiated by nutrient limitation at a high cell density. After the removal of nutrients, M. xanthus cells sense their nutritional status and cell density by monitoring the increase in the extracellular A signal concentration, and subsequently activate the A signal transduction pathway. Activation occurs only when the extracellular A signal concentration is within a specific intermediate range. Using a sensitive sensing mechanism, the A signal pathway directs the progression through the early stages of M. xanthus development. Our long term goals are to determine: 1) how the cells sense the extracellular A signal concentration, 2) how this information is transduced, resulting in a change in gene expression and 3) what is the connection between the change in gene expression and the complex behavioral response of multicellular fruiting body formation. To answer these questions we will characterize the regulatory network of the A signal transduction pathway. Several components of this circuitry have been identified, including the extracellular signal, A signal, and a specific response, an increase in expression of a gene, gene4521. A regulatory element has been identified which maps to the sasA (for suppressor of A signal) locus. The sasA locus is required for the progression through early development and differentially regulates the expression of distinct A signal-dependent genes. The focus of this research is to use classical and molecular genetic techniques, combined with protein biochemistry, to fully characterize the sasA locus and its encoded regulatory element. Molecular genetic approaches will be used to analyze the sasA locus structure by defining the physical limits of the locus, analyzing the transcripts, sequencing the region, identifying the open reading frame(s), and construction and characterizing a null mutation. To study the function of the sasA gene product we will determine if the sasA gene product directly or indirectly regulates gene4521 expression. First, the cis-acting regulatory elements controlling gene4521 expression will be determined by deletion analysis. Then, biochemical techniques will be used to test binding of purified sasA gene product to the gene4521 regulatory region. Any binding activity will be characterized in detail. If the sasA gene product appears to indirectly regulate gene4521 expression, biochemical techniques as well as genetic screens will be used to identify possible intermediate regulatory elements. In addition, genetic screens will be developed to identify other components of the A signal transduction pathway, which will likely include a receptor and additional transduction elements.